Method and compositions for promotion of wound treatment

ABSTRACT

The present invention comprises compositions and preparations for the promotion of wound healing in an animal. Methods for preparing the compositions as well as methods for using the compositions to achieve the promotion of wound healing, are also provided. The composition may comprise a dietary regimen or a therapeutic agent. These compositions include a wound healing promoting concentration of nucleotides. By way of example, such nucleotides may comprise RNA, adenine, uracil or a mixture thereof. The compositions can be prepared as suitable for oral, parenteral, intravenous or topical administration. Methods for using the preparation as a treatment to enhance the healing of an already existing wound or for use as a pretreatment regimen for animals in anticipation of surgery, are also disclosed.

This is a continuation-in-part of U.S. Ser. No. 08/309,958, filed Sep.21, 1994 now U.S. Pat. No. 5,712,256, Jan. 27, 1998, that is acontinuation of U.S. Ser. No. 08/086,346, filed Jun. 30, 1993,abandoned.

The United States government has a paid-up license in this invention andthe right in limited circumstances to require the patent owner tolicense others on reasonable terms as provided by the terms of Grant No.RO1-CA3 5492 awarded by National Institutes of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to techniques used in the promotion ofwound healing. In particular, compositions of matter that promote thehealing of wounds, methods of manufacture of wound healing promotingcompositions, and methods of treatment that promote wound healing areencompassed within the scope of the present invention.

2. Description of the Related Art

Animals, including human beings are susceptible to a barrage of normalcuts and scrapes, as well as to much more serious wounds that mayrequire medical attention.

Wounds may be the result of accidents or surgery. For the most part,such wounds heal at a fairly steady and slow rate, being affected bymany factors including the nature and site of the wound and thephysiological state of the animal.

The process of wound healing involves many complicated components.Immediately upon the injury insult, defense mechanisms inherent innormal body tissues are activated to restore continuity and tensilestrength. Wound healing then occurs in three distinct phases.

First, is the phase of acute inflammatory response. Body fluidscontaining plasma proteins, fibrin, antibodies and various blood cellsflow into the wound. Scab formation takes place and inflammation occurswithin a few hours. Also, at this stage, neutrophils,monocytemacrophages come into play. During this acute phase, the woundis solely dependent on the closure material contained in the scab forstrength.

Second, is the phase of fibroplasia. Here, via various enzymaticmechanisms, fibrin synthesis and accumulation takes place. This causesan increase in wound tensile strength and stimulation of fibroblastproliferation and growth. Fibroblasts secrete collagen, a fibrousprotein as part of connective tissue. Collagen deposition begins fromthe fifth day and results in rapid gain in tensile strength of thewound.

The third phase is the maturational process. Tensile strength continuesto increase from the cross-linking of collagen fibers. Deposition offibrous connective tissue causes scar formation.

Collagen production is vital for the wound healing process. Collagen isthe most prevalent protein in animals. It is an obligatory constituentof connective tissues and extra cellular matrices. Collagen networks inthe tissues are responsible for establishing and maintaining thephysical integrity of diverse extra cellular structures. Collagen, atmolecular level, is defined as a protein comprised of lengthy domains oftriple-helical confirmation. Collagenous scaffolding of extra cellularmatrix includes genetically distinct types of collagen. During thenormal wound repair, collagen neosynthesis and deposition of type IIIcollagen is demonstrated in the earliest phase, i.e. 24 hr to 48 hr,period. From that point, a significant increase in type I collagen isassociated with the mature wound fibroblasts and subsequent healingevents. Because of its important role in the wound healing process,collagen production is a measure of the rate and quality of woundhealing. As such, assays that measure collagen production are useful inexperimental models to study wound healing.

The healing process is very much organ and tissue-type dependent. Forexample, intestinal tissue is physiologically a rapidly self emphasizingtissue and unlike other organs in that it must constantly be repaired.Intestinal repair is an ongoing process necessary to maintain normalfunction of the intestines. There is an almost constant need for repairin the intestines, where injury arises from aberrations in the digestiveprocess or from ingested foods. In contrast to intestinal repair, the“wound healing” discussed in this application is caused by externalfactors of trauma and injury. Such sudden and external trauma injuryrequires intact and able host defense mechanisms.

The process of wound healing involves a complex system of local andremote (systemic) energy and substrate requirements and uses. Forexample, amino acids and sugars are needed as substrates for collagenand proteoglycan synthesis. Migration of fibroblasts andepithelial/endothelial cells during the wound healing process placesadditional systemic demands on the animal during the wound healingprocess. Wounded tissues have unique nutritional needs and physiologicalfeatures. Lymphocyte participation in wound healing has beendemonstrated (Peterson et al. (1987)). Alteration in the hosts T-celldependent immune response has also been shown to influence woundhealing. Cyclosporine and anti T-cell antibodies, both of whichinterfere with T-cell function, abrogate wound healing. Similarly,macrophages and their products are also involved in wound healing.Increased circulation usually results in rapid delivery of monocytes andPMN's to the wound site. This in turn results in the elimination ofbacterial contamination of the wound due to nonspecific killingmechanisms and also enhances the rate of wound healing. These variouscell types are synthesized by the bone marrow.

In many cases, the wound healing process proceeds very slowly,particularly in animals having limited energy stores or diets low inenergy substrate sources.

Purine and pyrimidine nucleotides are involved in almost all cellularprocesses and play a major role in structural, metabolic, energetic andregulatory functions. They make up the monomeric units of RNA and DNA;RNA synthesis is required for protein synthesis and DNA synthesis isrequired for growth and cell division. Adenosine triphosphate, anadenine nucleotide is the major source of chemical energy used inmetabolism, driving almost all cellular processes. Nucleotides arephysiological mediators in a number of metabolic processes. Cyclicadenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)regulate a large number of cellular events, and adenosine is importantin regulating blood blow and smooth-muscle activity. Guanosinetriphosphate (GTP) is involved in signal transduction, RNA structure,and microtubule formation. Many other nucleotides are involved inregulating other cellular processes. Nucleotides function as activatedintermediates in the synthesis of glycogen and glycoproteins; they arealso intermediates in the synthesis of phospholipids, and serve asmethyl and sulfate donors. They are structural components in a number ofcoenzyme that are crucial in many metabolic pathways, and they functionas allosteric effectors that control the regulatory steps of majormetabolic pathways.

Nucleotides consist of a nitrogenous base (either a purine or apyrimidine), a sugar, and one or more phosphate groups. The termnucleotide in the context of the title refers to the multiple forms inwhich purines and pyrimidines are found and does not imply a specificform of the compounds but all forms that contain purine and pyrimidinebases.

The major purine bases are adenine, guanine, hypoxanthine and xanthine.Uric acid is also found in significant levels. The major pyrimidinebases are uracil, thymine, and cytosine. Other pyrimidines and purinesare also present in smaller amounts and they have significant rolesparticularly in RNA structure and function.

The nucleotides are phosphoric acid esters of nucleosides in which thephosphoric acid is esterified to one of the free pentose hydroxylgroups. Nucleotides occur in free form in significant amounts in avariety of cell types. They are also formed on partial hydrolysis ofnucleic acids, particularly by the action of a class of enzymes callednucleases. Nucleotides containing 2-deoxy-D-ribose aredeoxyribonucleotides; those containing D-ribose are ribonucleotides. Anucleoside, which does not have a phosphate group, is formed from a baseand a pentose via a glycosidic bond between the N-1 nitrogen of apyrimidine or the N-8 of a purine and the C-1′ carbon of the pentose.The pentose is ribose or 2′-deoxyribose. The major function of the2′-deoxyribose nucleotides is in DNA. The ribonucleotides are themonomeric units of RNA but also serve in most other cellular andmetabolic functions of nucleotides. The phosphoryl group of nucleotidesis most commonly esterified to the C-1′ hydroxyl of the pentose. Incyclic nucleotides the phosphate is esterified to both the C-5′ and C-3hydroxyl groups. The number of phosphate groups attached is indicated bya mono-, di- or tri-designation. In the discussion and description ofthe claims the term nucleotide will be used generally to mean a sourceof preformed purines and/or pyrimidines in various forms including RNAas well as individual purines and/or pyrimidines as bases, nucleosidesor nucleotides. It does not generally (except as noted in specificexamples) imply that one form is required.

Since there are two or more free hydroxyl groups in nucleosides, thephosphate group of nucleotides can potentially occur in more than oneposition on the sugar ring. In the case of deoxyribonucleotides, thereare only two possible positions in 2-deoxyribose that can be esterifiedwith phosphoric acid, namely, the 3′ and 5′ positions. Both 3′- and5′-deoxyribonucleotides occur biologically. In the case ofribonucleotides, the phosphate group may be at the 2′, 3′, or 5′position; all 3 types of ribonucleotides have been found as hydrolysisproducts of RNA, depending on conditions. Cyclic monophosphates ofadenosine are also possible. However, the nucleotides that occur in thefree form in cells are predominantly those having the phosphate group inthe 5′ position, since the enzymatic reactions normally involved innucleic acid synthesis and breakdown in cells proceed via nucleoside5′-phosphates intermediates. Table 1 gives the nomenclature of the majorribonucleoside 5′-monophosphates (also called 5′-ribonucleotides) anddeoxyribonucleoside 5′-monophosphates (also called5′-deoxyribonucleotides). All the common ribonucleosides and2′-deoxyribonucleosides also occur in cells as the 5′-diphosphates andthe 5′-triphosphates, i.e., the 5′-pyrophosphoric and the5′-triphosphoric acid esters of the nucleosides.

TABLE 1 Nomenclature of bases, nucleosides, and nucleotidesRibonucleoside 5′-mono-, di-, Deoxyribonucleoside 5′- and triphosphatesmono, di-, and Base Ribonucleoside Abbreviations Deoxyribonucleosidetriphosphates Adenine (A) Adenosine AMP ADP ATP Deoxyadenosine dAMP dADPdATP Guanine (G) Guanosine GMP GDP GTP Deoxyguanosine dGMP dGDP dGTPUracil (U) Uridine UMP UDP UTP Cytosine (C) Cytidine GMP CDP CTPDeoxycytidine dCMP dCDP dCTP Thymine (T) Deoxythymidine dTMP dTDP dTTP

Purines and pyrimidines can be formed by de novo biosynthesis or salvageof preformed bases and interconversion to the desired compound. Almostall of the atoms in both bases are derived directly or indirectly fromamino acids. Phosphoribosylpyrophosphate (PRPP) serves as the pentosesource for both purine and pyrimidine biosynthesis and for salvage ofbases. PRPP is formed from ribose-5-phosphate. Deoxyribonucleotides aresubsequently formed from the ribonucleotides.

The pathway for purine biosynthesis consists of ten steps. The initialstep involving PRPP and glutamine condensation catalyzed by PRPPaminotransferase is likely the rate limiting step and is feed-backinhibited by AMP and GMP. IMP is the first purine formed and it isconverted to either AMP or GMP depending on cell needs. Regulationoccurs at these steps also. The monophosphates of both purines andpyrimidines are readily converted to di- and triphosphates by variouskinase enzymes using ATP as a phosphate source.

In pyrimidine biosynthesis, PRPP is not added until the intactpyrimidine is formed as orotic acid. OMP (orotidine-5′-monophosphate) isthe first pyrimidine formed but it functions in the cell only as aprecursor of other pyrimidines. UMP is formed from OMP and then CTP andTTP are derived from UMP. In eukaryotes regulation of pyrimidinesynthesis occurs primarily at carbamoyl phosphate synthesis withinhibition by pyrimidine nucleotides and activation by purinenucleotides.

Deoxyribonucleotide synthesis is catalyzed by ribonucleotide reductase,an enzyme that converts both purine and pyrimidines to their deoxyriboseforms. The reductase is controlled in a complex manner by bothsubstrates and product to allow synthesis of equimolar levels of thevarious deoxyribonucleotides. Since the deoxynucleotides are used onlyfor DNA synthesis the levels of the purine and pyrimidine need to beequal. Thymidine triphosphate is then formed as the monophosphate fromdeoxy-UMP. The levels of the deoxyribonucleotides are typically in therange of 2-60 mM while ribonucleotides are typically much higher withATP concentration in the range of 2-10 mM and other ribonucleotides from0.05-2 mM. Di- and monophosphates are typically lower than thetriphosphates. Levels of both ribo- and deoxyribonucleotides will varyconsiderably depending on the phase of the cell cycle and under variousmetabolic conditions.

In primates uric acid is the end product of purine catabolism whileother species can convert it to more soluble forms. The end products ofpyrimidine catabolism are β-alanine and β-amino isobutyrate which areboth soluble and easily excreted. Less is known about pyrimidinecatabolism since no clinical effects of the end products occur. Thecatabolic pathways operate in the digestive system converting DNA andRNA and free nucleotides to nucleosides and free bases. Pyrimidine basesand nucleosides are taken up and readily incorporated into tissues.Dietary nucleotides appear to be important in support of cellularmetabolism particularly in rapidly dividing tissues such as lymphoidcells and the intestine.

The uptake of purines and pyrimidines from the intestine and cellularturnover of nucleotides particularly from mRNA provides preformed basesthat avoid the metabolic cost of de novo biosynthesis. Synthesis of bothpurines and pyrimidine consumes a significant amount of energy. It isimportant to note that role of amino acids in nucleotide synthesis andthe salvage of dietary and cellular sources of nucleotides. A balanceexists between these different pathways affording proper levels ofnucleotides in cells with minimal metabolic expense.

The usefulness of dietary nucleotides in certain medical contexts isdocumented. The instant inventors and others have described thepotential role of dietary nucleotides in several contexts. For example,dietary nucleotides are required for maintenance and recovery of hostimmune response (Van Buren et al. (1983) and Rudolph et al. (1990)). Ithas also been shown that there is increased activity of Lyt1+, IL2-R+and Mac1+ cells in the tissues responding to alloimmune challenge (VanBuren et al. (1985) and Kulkarni et al. (1988)). Nucleotidesupplementation has also been shown to provide an increase in bothimmunohemopoiesis (Kulkarni et al. (1992)) and resistance to infectiousmicroorganisms (Kulkarni et al. (1986)). Nucleotide supplementation hasalso been described as reversing immunosuppression induced by proteinstarvation. (Pizzini et al. (1990)).

Several research groups have published works concerning the relationshipof nucleotides to immune system functioning. Van Buren et al. (1985)relates to the role of dietary nucleotides in the processes ofrecognition of and sensitivity to foreign antigens and in lymphocyteproliferation to alloantigen or lectin stimulation. The presentinventors have also described the importance of dietary sources ofpyrimidines and purines, such as those in nucleic acids, in immunefunction and on gastrointestinal function. (Rudolph et al. (1990))Normal cellular immune response has therefore been postulated to requirea source of preformed nucleotides. The authors conclude that dietarysources of nucleotides are important to support optimal growth andfunction of metabolically active cells such as lymphocytes, macrophagesand intestinal cells.

The role of dietary nucleotides in the immune response is furtherexamined in Pizzini et al. (1990). In this series of studies, nucleotiderestriction was tested using both a starvation malnutrition and aprotein malnutrition in vivo model. Animals in the starvationmalnutrition study receiving a diet supplemented with yeast RNA prior tothe period of starvation (5 days) reportedly demonstrated an increase inspontaneous concanavalin A and phytohemagglutinin-stimulatedblastogenesis in in vitro assays. In protein malnutrition studies, thereturn to any of the examined diets (chow diet, nucleotide-free diet, ornucleotide free diet supplemented with 0.25% yeast RNA) reportedlyresulted in restoration of body weight, while only the RNA-supplementedand chow diets restored popliteal lymph node immune reactivity.

The usefulness of nucleotides in the repair or regeneration ofintestinal gut cells in infants was the basis of the U.S. Pat. No.4,994,442. This patent relates to a milk and non-milk based infantformula that includes nucleosides and/or nucleotides. As previouslystated, this process of intestinal repair is continual andphysiologically distinct from wound healing in response to trauma orinsult, which is the goal of the present invention.

The role of dietary nucleotides in preventing the onset of infection hasalso been studied. In Kulkami et al. (1986), the present inventorspresent data relating to the role of dietary nucleotides (for example,dietary adenine, uracil or RNA) in maintaining animal resistance toStaphylococcus aureus. Fanslow et al. (1988) examines the relationshipbetween dietary nucleotides and animal susceptibility to candidiasis.Studt et al. (U.S. Pat. No. 4,486,439) relates to a method for treatingcoccidial infections employing a formulation that includes, among otheringredients, 2-pyrimidine, 4-pyrimidine, 5-pyrimidine, 6-pyrimidine,2-purine, 6-purine, 8-purine or 9-purine.

Dietary nucleotides have been implicated as having a role in relation todelayed cutaneous hypersensitivity (Kulkami et al. 1987) and in thefatty acid composition of erythrocyte membrane lipids in infants(DeLucchi et al. 1987).

Gil et al. (U.S. Pat. No. 5,066,500) relates primarily to a non-milkbased infant formula that includes amino acids and is enriched withnucleotides and/or nucleosides (at least one of uridine,uridine-phosphate, guanosine or guanosine phosphate, adenosine oradenosine phosphate, cytidine or cytidine phosphate, inosine or inosinephosphate, or mixtures thereof). Examples are also provided of definedcomposition dietary supplements for adults suffering from suchnon-trauma or insult problems as energy-protein malnutrition,hypercatabolism, malabsorption-malnutrition syndromes, severehomeopathy, or chronic hematopathy. However, these formulations are notused to stimulate the immune system. Gut intestinal cell turnover is anormal, physiologic process, in which the inflammatory response plays norole. Healing of a traumatic wound, on the other hand, requires aninflammatory response as a necessary first step in wound healing.

A respiratory enzyme booster tablet that includes a combination ofdiphosphopyridine nucleotide, nicotinamide, adenosine-5-monophosphateand a carrier has been described in the Case patent (U.S. Pat. No.4,308,257). The compound functions as a co-enzyme that acts in thecellular respiration process. An injectable treatment that includesdiphosphopyridine nucleotide is also described. The use of a nucleotidecompound in the absence of other ingredients however, has not beendescribed, nor suggested as a potentially useful therapeutic agent.Also, these tablets are specifically designed to increase the rate ofcellular respiration, a phenomenon that occurs in all cells. Theseformulations do not appear to play a role in the enhancement of collagenformation or wound-healing.

The Guari patent (EP No. 85,084, 1983) relates to a wound-healing andantiviral preparation which includes a dialysis concentrate ofdeproteinated calf s blood and a member of a very specific class offuranosylated, uracil derived compounds. These ingredients reportedlyact “synergistically” to provide the described physiological effects.

The idea and process of nutritional therapeutic approach would have noside effects (toxic or untoward) as shown many times by thepharmacologic or chemotherapeutic interventions. Injury or traumainduced stress causes sudden loss of body fluids and nutrients, propernutritional repletion can improve these losses. The effects may besustaining and long term rather than symptomatic quick-fix afforded byother means. Nutritional modulation may help and improve the endogenousphysiologic process in order to combat the wound-related trauma.

In reviewing the known related art, it becomes apparent that there hasbeen no suggestion of the usefulness of nucleotides as pharmacologicallyactive agents in the relatively complex, processes of wound healing. Forexample, the specific events important in wound healing of collagenformation, fibroblast proliferation and restoration and maintenance ofhost immune response have not been described or suggested to be enhancedthrough dietary supplementation with nucleotides.

Normal wound healing can be impaired by chronic infection, proteinmalnutrition, poor blood supply, vitamin deficiencies, previousradiation exposure, diabetes mellitus, corticosteroid therapy anddeficiencies in the components of the host wound response. Obviously,many of these conditions are more likely to cause problems the longer awound takes to heal. Additionally, escalating health care costs indicatea need for methods that promote wound healing. Therefore, any proceduresthat would aid in wound healing would be welcomed in the medical field.

SUMMARY OF THE INVENTION

The problems associated with wound healing are in part remedied by thecompositions and methods of the present invention. The inventors havefound that wound healing can be greatly enhanced by the inclusion ofnucleotides and/or substances that include essential nucleotides, suchas RNA, DNA, oligonucleotides, purine and pyrimidine bases, or any othersource in a pharmaceutical preparation. Dietary nucleotides are alsoproposed by the present inventors to be useful in pretreatment regimensto enhance wound healing in, for example, surgery patients. Theinvention provides for the use of nucleotides in concentrationseffective to promote wound healing. Great utility is realized with thedescribed compositions and methods in enhancing the rate of woundhealing, and the wound healing process in general. A more rapid woundhealing process also is anticipated to reduce recovery time. Concomitantbenefits would also include a reduction in medical costs and treatment,time away from work, and the incidence and severity of infection.

The inventors have shown that dietary nucleotides modulate various hostimmune parameters, especially in protein-malnutrition induced stress,nucleotide supplemented diets improve rapidly the host immune system.This has been shown by various in vivo assays examining the immunologiccapacity as well, as evidenced by an increased resistance to sepsisobserved by the present inventors. It is felt that utilization ofexogenously supplied nucleotides by T-lymphocytes and macrophages of thebody's immune system is independent of provision of dietary protein. Aunique quality of dietary nucleotides heretofore undescribed for anyother nutritional substrate improves systemic host immune response, bothspecific and nonspecific, is therefore provided by the presentinvention. Such a boost of the immune response then, in turn, respondsto the body's requirements for alleviating insults. Such insults wouldinclude trauma, injury, either external or internal that would requireimmediate repair in order to maintain proper body physiology andfunction. The wound models described in this application examine suchcases of injury.

The present invention contemplates a therapeutic agent for the promotionof wound healing. In one preferred embodiment, the therapeutic agentcomprises a therapeutically effective concentration of nucleotides(i.e., effective to promote wound healing) in a pharmacologicallyacceptable carrier. The nucleotides contained in the “active compound”of the therapeutic agent may comprise RNA, adenine, uridine, any of thecompounds contained in Table 1, or a combination thereof. In somepreferred embodiments of this invention, the nucleotide componentcomprises RNA, adenine, uridine, inosine or a mixture thereof. Whilealmost any level of nucleotide administration is expected to be ofbenefit in the wound healing process, it is anticipated thatconcentrations of about 0.10% to 0.50% (ranging from 0.00034 g/kg bodywt/day to 0.17 g/kg body wt/day) will be particularly useful. Theseconcentrations are for purines and pyrimidines in the form ofnucleotides in the pure chemical sense, i.e. with a phosphate group. Ifnucleosides are administered, the concentrations will range from 0.00022g/kg/day-0.12 g/kg/day, since nucleotides do not contain the weight of aphosphate group. As mentioned previously, the use of the term“nucleotide” elsewhere in the application means both nucleotide andnucleoside forms of the purines and pyrimidines. In the claims, a claimto concentrations of nucleotides, (i.e. 0.00034-0.17 g/kg/day)encompasses the equivalent amount of nucleoside (i.e. 0.00022-0.12g/kg/day). A concentration of about 0.25% represents a most preferredembodiment of the present invention.

A decided practical advantage is that the nucleotides that comprise theactive compounds of the present invention may be administered as adietary supplement in any convenient manner, such as by the oral,intravenous, intramuscular, or subcutaneous routes. The dosage regimenof this dietary therapy may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The nucleotides may be orally administered, for example, with an inertdiluent or with an assimilable edible carrier, or they may be enclosedin hard or soft shell gelatin capsule, or they may be compressed intotablets, or they may be incorporated directly with the food of the diet.Since there is no disagreeable taste to nucleotides, they could besupplied in a powdered form to be mixed with food by the patient. Fororal therapeutic administration, the active compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should containsufficient active compound so as to administer at least 0.00034 g/kgbody weight active compound per day. The percentage of the compositionsand preparations may, of course, be varied according to the specifics ofa therapeutic situation. The amount of active compounds in suchtherapeutically useful compositions should be such that a suitabledosage will be obtained when a compositions is administered in asuitable way.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup or elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

The nucleotides may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. Finally, the nucleotides could be supplied topicallywith a gel, powder, salve, or patch.

As used herein, “pharmacologically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated.

Of course, the nucleotide active compounds of the present invention maybe administered by any of the other numerous techniques known to thoseof skill in the art. (For a reference on these techniques seeRemington's Pharmaceutical Science 18^(th) Edition, (1990)) which isspecifically incorporated herein in pertinent part for this purpose).Supplementary active ingredients can also be incorporated into thecompositions.

The present invention also is proposed to provide a dietary supplementfor the promotion of wound healing. This regimen may comprise aconcentration of nucleotides therapeutically effective to promote woundhealing in a pharmacologically acceptable carrier solution. The dietarysupplement prepared in any suitable form, for example as a liquidsuitable for injection, parenteral administration, or oraladministration or as powder suitable for mixing with food or a beverage,e.g., as tablets or capsules. It is contemplated that the dietaryregimen of the invention may be administered to an animal either as apretreatment in anticipation of surgery or after a wound has occurred toboth hasten and enhance the quality of wound healing.

The present invention also includes methods for promoting wound healingin animals. These methods comprise preparing a composition ofnucleotides effective to promote wound healing and treating an animalwith an effective concentration of the composition. These methods can beof use in treating a wound that presently exists or a wound that mayexist in the future, for example, in the case of a scheduled surgery.Thus, the present formulations may be used as part of a pretreatmentplan that would provide a heightened level of nucleotides in the animalprior to surgery that will in turn enhance both the healing process andthe rate at which the wound is healed.

It is projected that it will be beneficial to place many, if not all,surgery patients on a nucleotide pre-treatment regimen to promote themore rapid healing of incisions, etc., that occur during surgery. Forexample, a patient would be given a nucleotide concentration effectiveto promote wound healing in any of the previously suggested forms fromthe time of the diagnosis of the need of surgery until a prescribed timepost-surgery when the wound has healed satisfactorily. It is projectedthat nucleotide treatment can be done for an appropriate period prior tosurgery. This period may be quite short in a stressed person, but couldbe as long as a number of weeks.

The present invention also contemplates methods of enhancing the rate ofwound healing with the administration of a therapeutically effectiveconcentration of nucleotides to a wounded animal. Such an enhancementwill most times also involve an increase in the collagen content of awounded area.

The present invention contemplates methods encompassing a pretreatmentregimen for enhancing the rate of wound healing in an animal that is toundergo surgery. These methods comprise the administration of atherapeutically effective concentration of nucleotides in apharmacologically acceptable carrier to an animal. A most preferredembodiment the pretreatment method is expected to involve pretreatmentfor up to around 4 weeks prior to surgery. However, benefits of thismethod can be expected with shorter lengths of pretreatment. It isanticipated that the preferred embodiments of these pretreatment methodswill comprise as active compounds RNA, adenine, uracil or a mixturethereof as the source of nucleotides. Of course, those of skill in theart will understand that other sources of nucleotides will be useful asactive compounds in this invention.

The present invention also contemplates a method of preparing atherapeutic agent for the promotion of wound healing comprising placinga wound healing promoting concentration of nucleotides in apharmacologically acceptable carrier solution. The carrier shouldprovide an adequate means for delivering the nucleotides to an animal inneed thereof. This preparation could be in solid form (such as inpowdered capsule or tablet form) or in a liquid form (suitable forinjection, parenteral or oral administration).

The present invention therefore provides improved therapeutic agents forwound healing, methods for the preparation of these therapeutic agents,and methods for the promotion and enhancement of the rate of woundhealing. These compositions and methods are anticipated to provide for amore rapid and complete wound healing in animals. As wound healing isthe most catastrophic and costly problem associated with surgery, theadvantages of reduced medical complications associated with the healingprocess and improved quality of wound healing will provide a significantadvancement in patient post-surgical clinical management. These andother advantages of the present invention will be further appreciatedfrom the detailed description provided below.

One embodiment of the present invention involves a method for promotingwound healing in a diabetic subject. This involves preparing a dietarycomposition supplemented with RNA, adenine, uracil, inosine or adenosinein an amount effective for promoting wound healing and feeding thediabetic subject the composition. Another important component of thepresent invention is a method for promoting wound healing in an animalsubject to protein-deficient nutrition. This involves preparing adietary composition supplemented with RNA, adenine, uracil, inosine oradenosine in an amount effective for promoting wound healing and feedingthe protein-deficient animal with the composition. This method isfurther enhanced if the protein or amino acid source is also fed to theanimal at this time.

In one important aspect, the present invention may involve preparing asterile composition comprising, RNA, adenine, uracil, inosine oradenosine in an amount effective to promote wound healing and topicallytreating the wound of an animal with the composition. Another importantembodiment of the present invention for the promotion of wound healingcomprises obtaining a crosslinked collagen mesh. This embodiment furtherinvolves obtaining a composition supplemented with RNA, adenine, uracil,inosine or adenosine in an amount effective for promoting wound healingthen emplacing the crosslinked collagen mesh on the wound and treatingthe animal with the composition. The treatment may be topical orenteral, in any manner such that the wound area has increased nucleotideconcentrations. The compositions of the present invention may bedesigned to be intravascularly administered. For example, when a patientis on a parenteral feed, it might be expected that the feed wasdeficient in amino acids. Thus a parenteral feed comprising thenucleotide compositions described above as well as additional aminoacids or proteins may be substituted. Topical administration of thenucleotides and possibly protein or amino acids should also beeffective.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Wound collagen content of a wound as a function ofhydroxyproline (OHP) in mice fed one of the following diets: normal (F),nucleotide free (NF), nucleotide free supplemented with adenine (NFA),nucleotide free with RNA (NFR), nucleotide free with uracil (NFU).

FIGS. 2 and 3 show mice wounds at days 0, 2 and 5.

FIG. 4 shows the wounded mice after two weeks

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides preparations and methods of using thepreparations for the enhancement of the quality and rate of woundhealing. Methods for preparing the various formulations are alsoprovided. The preparations/formulas of the present invention maycomprise a dietary regimen or a therapeutic agent. As a therapeuticagent, the invention comprises a wound healing promoting concentrationof nucleotides together in a pharmaceutical acceptable carrier. Thetherapeutic agents of the invention may be delivered to an organismthrough any of a number of routes with equal therapeutic efficiency. Themethods of the present invention may vary in the means of deliverychosen, the type of organism treated, and the time-frame of thetreatment relative to the time of the wounding. There are also a varietyof methods of preparing the preparations/formulations encompassed withinthe contemplated scope of the present invention.

The following examples are intended to illustrate the practice of thepresent invention and are not intended to be limiting. Although theinvention is demonstrated with an RNA nucleotide, other nucleotideshaving would healing promoting activity may be used in a similarfashion.

MATERIALS AND METHODS

Animals and Diets

Balb/c mice (Jax Labs), 8 weeks old, are typically used in thesestudies. Custom made diets from a can be obtained from a commercialfacility such as Purina Test Diets or produced in the lab.

Animals are maintained on specific diets designed to test the effects ofnucleotides on wound healing. An example of one such experimental dietregime, which could be fed diets prior to implantation of woundcylinders could be: Formula chow (F), Basal diet (nucleotide free) (NF),NF supplemented with 0.25% yeast RNA (NFR), or 0.06% uracil (NFU).

A protein starvation model can also be used in conjunction with thesestudies. In such a protocol, animals are implanted with wound cylindersand on the same day mice are placed on one of the following diets:Protein free (PF), PF supplemented with 0.25% yeast RNA (PFR), and PFsupplemented with protein (NF). Mice are maintained on these diets untilthe day of sacrifice i.e. day 14 from implantation.

Measurement of Collagen Synthesis in Wound Healing

Studies to test the hypothesis that dietary nucleotides are required forcollagen synthesis in wound healing can be assessed using the method ofGoodson and Hunt 1982). In this protocol, a 2 cm longpolytetrafluoroethylene (PTFE) (3 mm lumen, 90 m pore size) wouldcylinder (WL Gore Associates) is placed subcutaneously in the orsalmidline of each animal while each animal is under general anesthesiapentobarbital). Cylinders are then injected with 0.2 ml of phosphatebuffered saline. On day 14 or another suitable time, animals aresacrificed and intact wound cylinders retrieved and frozen untilanalysis for hydroxyproline content and collagenase assay.

The hydroxyproline assay is performed as follows. A portion of woundcylinder implant from each animal is used for preparing acidhydrolysates for measuring hydroxyproline concentrations in this assayusing the method of Woessner (1961), which reference is specificallyincorporated herein by reference for this purpose. The hydroxyprolineconcentration within each implant, a measure of collagen content can bedetermined spectrophotometrically and expressed as micrograms ofhydroxyproline per centimeter of PTFE implant.

Collagenase plays a significant role in wound healing. In the woundrepair process, collagen synthesis and accumulation is important.Careful and appropriate degradation of collagen is very important inwound healing repair and tissue formation. The collagen fibril, formedas required by aggregation of collagen monomers, is extremely effectivestructural element for maintaining the integrity of the newly formedconnective tissue. These fibrils are physically stable up to 50° C. andare chemically resistant. Fibrillar collagen is essentially insolubleunder normal physiological conditions. It is resistant to degradationaction of a wide range of naturally occurring proteolytic enzymes.However, host cells have the ability for endogenous production ofspecific enzymes—collagenases—which act primarily on collagen. Theseenzymes, by proteolytic cleavage denature each of collagen fibers. Thusfor appropriate wound healing and formation of repair-tissue and itsstructural integrity, endogenous production of collagenase is essential.The measurement of collagenase in wound tissue is an indicator of woundhealing strength. For this assay, one can use a collagenase assay systemsuch as the one available from New England Nuclear (NEN-cat# NEK016),employing 3H-collagen. Collagenolytic activity is monitored with a highspecific activity substrate by quantitating the production of solubleradioactive fragments, which are readily separated from undigestedcollagen fibrils by centrifugation.

The hydroxyproline assay and the collagenase assays, as discussed above,lend themselves to both the nucleotide supplemented and the proteinstarved animal models.

Colonic Tensile Strength and Wound Healing with Nucleotides

Studies can be conducted to determine whether dietary nucleotidesimprove colonic wound bursting (tensile) strength. The colonic tensilestrength model relates to the type of tissue that is highly dependent onrapid fibroblastic regeneration and formation of a strong matrix. Thismeans rapid accumulation of collagen fibers in the wound. The reparativecollagen and its fibers deposition attributes to the strength of thetissue which is measured by the model described by Nelsen and Anders(1966), referred in the reference section and specifically incorporatedherein in pertinent part.

This method involves testing the bursting strength of intestinalanastomoses by distention with either air or water. The traction methodof testing bursting strength can also be employed. Lengths of ilealsmall bowel, usually around 6-8 cm, are isolated and divided in themiddle with proper surgical techniques and resutured as end to endanastomosis (using appropriate sutures). These surgical procedures andtechniques should be identical in all the animals involved in aparticular study, and is a general surgical technique well known tothose of skill in the art. On post-operative day 14, or at anothersuitable time, the animals are sacrificed and the operative area ofileal gut, with the anastomosis in the center, removed for the burstingstrength evaluation. All the segments are typically adjusted to anidentical collapsed length, attached at both ends to grooved rubberstoppers and securely tied with cotton tapes. Air is removed and thesegment is then filled with either air or water by an infusion pumpattached to one end of the segment through the stopper. All tests willbe carried out on a horizontal plate and with one end free to moveduring inflation. Continuous monitoring of the pressure and volume ismaintained until bursting. Results are then calculated for all dietarygroups and compared.

EXAMPLE 1 Nucleotides from RNA Enhance Wound Healing In Vivo

The present example demonstrates the utility of the present inventionwith dietary nucleotides in promoting more rapid on wound healing invivo. Yeast RNA is employed as an example of the particular nucleotidesthat may be employed in the practice of the present invention.Nucleotide supplementation is demonstrated to be beneficial for woundhealing. The present example also demonstrates the utility of theinvention for the promotion of wound healing, and for the promotion ofmore rapid wound healing, in humans. Dietary formulations andpreparations for enhancing the wound-healing process and the rate ofwound healing are also provided in the present example.

Wound healing was assessed by hydroxyproline (OHP) measurements in thePTFE matrix of a wound cylinder in a mouse model. OHP level within animplanted wound cylinder of PTFE as indicative of wound healing is anestablished model for the examination of the wound healing process.Briefly, fifteen Balb/C mice were divided in three groups (5 per group).Each group received one of the following diets and water ad libitum:F-Formula rodent chow, NF-nucleotide-free basal diet (Purina) and NFR-NFdiet supplemented with 0.25% yeast RNA (U.S. Biochemicals). Basal dietis composed of casein (21%) as source of protein. This source of proteinis unlike chow diet in that the standard chow diet has 23.5% proteinfrom corn, soybean, fish meal, meat, bone meal and milk. Basal diet ismade isonitrogenous and isocaloric with chow. Fifteen percent sugar isadded to make it isocaloric by adding carbohydrate. Basal diet does notcontain any purines or pyrimidines.

All animals received these diets 30 days prior to wound cylinderinstallation. PTFE wound cylinders were placed subcutaneously in thedorsal midline of each animal under general anesthesia. All animals werecontinued on their respective diets during the post-operative phase ofthe study. On the 10^(th) post-operative day, all animals wereeuthanized and the wound cylinders were removed for analysis and frozenuntil assayed. Wound cylinders were analyzed for hydroxyproline (OHP)content using the previously described method of Woessner. The data fromthis study is shown in Table 2.

TABLE 2 Wound Hydroxyproline Content in Various Dietary Groups mg OHP/cmPTFE Diet Group mean + sem F 8.95 + 0.67 NF 9.19 + 0.69 NFR 14.84 +1.00* * = significantly different from controls

p<0.001 vs NF,F

Five animals per group, 2 readings per animal.

OHP-Hydroxyproline

The data of Table 2 demonstrate a significantly lower concentration ofwound hydroxyproline in control animals as compared to animalsmaintained on an RNA-supplemented regimen. Hydroxyproline content in thewound tissue is an experimental indicator of the process of wound repair(Woessner and Hunt et al. 1991) Hydroxyproline is an indicator ofcollagen measurement. The collagenase activity measurement relates tocollagen content at the wound site. Mauch et al. (1989) have shown thatincreased collagenase gene expression in the wound tissue is inverselyproportional to the collagen content. Therefore, increased collagenaseactivity indicates ongoing degradation of collagen in the matrix andconsequently the presence of poor wound healing at an injured site.

As the data in Table 2 demonstrate, the diet containing RNA resulted ina statistically significant increase in OHP at the wound site, ascompared to OHP content in nucleotide free diet and standard dietmaintained animals. Increased OHP content provides a measure of collagenas the wound site, the data demonstrate that a nucleotide-enriched dietenhances collagen content at the wound site, thus indicating an enhancedamount of collagen available at the site for wound repair. A wound isexpected to heal more quickly where more collagen is available.

EXAMPLE 2 Adenine, Uracil and RNA enhance Wound Healing In Vivo

The present example is provided to demonstrate the utility of theinvention using adenine as a dietary source of nucleotide in the woundhealing promoting formulation of the invention.

Twenty-five Balb/C mice were divided into five test groups (n=5) andhoused in groups in the animal care facilities. Each group received oneof the following diets ad libertum: F—normal mouse chow, NF—nucleotidefree diet, NFR—yeast RNA supplemented diet, NFA—adenine supplementeddiet, and NFU—uracil supplemented diet. All groups received these dietsfor at least 30 days prior to wound cylinder implantation. All animalsreceived water ad libitum.

Polytetrafluorethylene (PTFE, Impra Vascular Graft, 3 mm lumen, 190 mmpore size) wound cylinders (1.5 cm long) were placed subcutaneously inthe dorsal midline of each animal under general anesthesia(Methoxyflurane, inhalation). All animals were continued on their testdiets. On the tenth post-operative day, all animals were euthanized bycervical dislocation and the would cylinders were removed for analysis.

Wound cylinders were analyzed for hydroxyproline (OHP) content using themethod of Woessner. Briefly, approximately 1 cm segments of the woundcylinders were hydrolyzed in 0.5 ml 6 N HCl for 3 hours at 130 C.Samples were cooled and 0.5 ml aliquots were neutralized with theappropriate volume of 1 N NaOH. Each sample was diluted to 5.25 ml withH₂O in order to reduce the total NaCl concentration to below 0.4 M. Eachsample was then reacted as described by Woessner.

FIG. 1 reports the results of this study. Adenine, RNA, and uracil allincreased the amount of OHP/cm PTFE over both the nucleotide free andthe non-supplemented diets. The increases seen for both adenine and RNAwere proven to be significant over the nucleotide free diet, while theincrease for the adenine supplemented diet was seen to be statisticallysignificant over the non-supplemented diet as well. In fact, the adeninesupplemented diet increased the amount of OHP in the wound by close totwo times.

PROPHETIC EXAMPLE 3 Preparation of Nucleotide-Containing TherapeuticComposition for Humans

The present example is provided to detail the preparation of anucleotide-enriched composition suitable for administration to humans.These compositions can contain any combination of nucleotides. However,the inventors have made preliminary observations suggesting thatcombinations comprising purines and pyrimidines or simply pyrimidinesalone may work better than those containing purines alone. Thiscomposition can be used as either a diet supplement or, with suitableadditions of nutrients, as an enteral or parenteral diet.

For example, a nucleotide-enriched liquid could contain about 0.25% RNA,dissolved in water or another suitable liquid. To make such apreparation, one mixes 2.5 g of RNA (from yeast or another source) witha liter of diluent. It, of course, may be necessary to add additionalingredients to place the liquid in a suitable form for feeding to apatient such as a semi-solid custard or soup.

The nucleotide should be administered at a level of from 0.00034-0.17g/kg body weight/day. Any concentration of nucleotides that willeffectively administer that amount of nucleotides in the particularcomposition being formulated will be useful. For example, a tablet willlikely need a much higher percentage concentration of nucleotides than acustard or other food to administer the required dose.

PROPHETIC EXAMPLE 4 Proposed Methods for Promoting/Enhancing the Rate ofWound Healing in Humans

The present prophetic example is provided to outline a proposed methodswhereby the nucleotide regimens of the invention may be used in thetreatment of humans for the promotion/enhancement of wound healing.

Examples of use of RNA or nucleotide sources as a dietary or topicalsubstrate for enhancing wound healing would include some of thefollowing clinical uses. Note that in the following example, RNA can besubstituted for similar levels of purines and/or pyrimidines.

A. RNA in a dose ranging from 0.00034 gm/kg/day to 0.17 gm/kg/day couldbe provided as part of a complete liquid diet with appropriate caloriesand protein in the same diet. This diet could be consumed as the soledietary source or a supplement to a patient who was eating. In a patientwhose intestinal function was adequate but who was not capable ofeating, the diet could be administered by means of a tube into thestomach or intestinal tract. The major difference between this treatmentand other enteral diets is that the RNA would be specifically enhancewound healing in a patient after an injury or after a surgicalprocedure.

B. RNA in a dose ranging from 0.00034 gm/kg/day to 0.17 gm/kg/day couldbe provided as an enteral supplement. The supplement could be mixed inwater and drunk, or could be administered by an enteral feeding tube. Ifthe patient were taking some food orally, the RNA supplemented could bemixed in custards, gelatin, or soups. Since RNA is virtually tasteless,no artificial flavorings would be required. The advantage of thisformulation is that the patient could have very little intestinalfunction, and yet could assimilate a low volume of fluid containing therequired dose of RNA. Thus, even after major gastrointestinal surgery,when most of the calories and protein might be administeredintravenously, an oral or enteral RNA supplement to enhance woundhealing could be provided.

C. Nucleobases most likely would be administered as nucleosides forparenteral use. This is due to the ionic nature of nucleotides whichmight impede transport of these substrates across cell membranes. Thedose of pyrimidine nucleosides would range from 0.00022 gm/kg/day to0.12 gm/kg/day delivered parenterally (these concentrations differ fromthe concentrations of nucleotides only because the nucleosides do nothave a phosphate group to add to their weight). The dose of purinenucleosides would range from 0.00022 gm/kg/day to 0.12 gm/kg/day withthe precaution that inosine should be substituted from adenosine athigher doses, due to pharmacological effects of adenosine. Specifically,adenosine has been recognized as a participant in the regulation ofcoronary, cerebral, skeletal and renal blood flows. Therefore, it may bedesirable to avoid large doses of adenosine. For a discussion ofadenosine's pharmacological effects, please see Belardinelli et al.(1989). These substrates could be administered with total parenteralnutrition or as additives to 5% dextrose or 0.9 M saline solutionsadministered by peripheral intravenous lines. Depending on the localinflammatory response, these substrates, could also be injectedsubcutaneously.

D. RNA or nucleosides can be administered topically via salve, anointment, an impregnated dressing, a sustained release patch, or as apowder. In this application the substrate can be applied directly to thewound to induce enhanced healing. The dose will range from 0.5 mM to 100mM.

EXAMPLE 5 Topical Applications for Wound Healing in Mice

Balb/c mice were divided into three dietary groups (F=normal mouse chow;NF=nucleotide free mouse chow; and NFR=nucleotide free mouse chowsupplemented with yeast RNA). Each group was fed before initiation ofwounds. After 4 weeks of feeding, a 4mm punch wound was created on thedorsal surface of each mouse, using a 4mm biopsy punch. The wounds werecoated with four different topical agents: 1) saline; 2) uracil {100 μMconcentration); 3) saline+CCM; and 4) Uracil+CCM (CCM=crosslinkedcollagen mesh). CCM is ‘Collagen Permeable Membrane’ from AccurateChemical & Scientific Corporation, catalog no. YIC 152299. Four mm sizecircles were soaked with appropriate nucleotide or saline solution (4mmpunch wounds) and used to dress the wounds.

Twelve groups were formed from these combinations (n=3). Mice werecontinued on their respective diets. Wounds were observed visually andphotographed at four different times during the experimental period oftwo weeks.

A record was made of the progressive contraction of the wounds and areaamong the various dietary and treatment groups. Two blinded observationsand results are summarized in Table 4.

TABLE 4 Diet Topical agent Relative efficacy F saline minimal F uracilmoderate F saline + CCM moderate F uracil + CCM significant NF salineminimal NF uracil minimal NF saline + CCM moderate NF uracil + CCMmoderate NFR saline moderate NFR uracil moderate NFR saline + CCMsignificant* NFR uracil + CCM significant* *With the methods at hand itwas difficult to distinguish the observations between the last twogroups of NFR fed mice.

These experiments were repeated using a 100 μM concentration of inosinein place of uracil as the topical agent. The results yielded nosignificant differences from that which was seen with the uracil.

EXAMPLE 6 Diabetic Mice

Rats were made diabetic by intraperitoneal inoculation of Streptozotocin(55mg/kg body weight). On day 1, if the glucose levels were below 250mg/dl, then they were reinjected. Only two animals required the seconddose. The readings were low on day 0, because the animals were fastedovernight before the induction of diabetes. Subsequent readings were onanimals after a 6 hour fast. Table 5 shows resultant blood glucoselevels.

TABLE 5 Glucose Levels in Diabetic & Non-Diabetic Dietary Rats Glucoselevel in mg/dl, mean ± sem Diet Day 0 Day 1 Day 14 Diabetic 75.7 ± 3.61306.7 ± 24.21 363.0 ± 22.31 Nondiabetic 62.9 ± 3.33 131.4 ± 12.68 139.0± 1.75  p value 0.0142* 0.0001* 0.0001* * = significant at 95%

EXAMPLE 7 Body Weights for Diabetic and Non-Diabetic Rats

The diabetic and nondiabetic rats were divided into two groups of diets:NF (n=14) and NFR (n=15). Each dietary group was divided into twogroups: Diabetic (NF:n=7, NFR:n=8); Non-diabetic (NF:n=7, NFR:n=7). Theresults and body weight for rats on each diet are shown in Table 6.

TABLE 6 Body Weights in Diabetic and Non-Diabetic Punch Wound Rats inDietary Groups Body weights in grams, mean ± sem Diet Day 0 Day 5 Day 8Day 15 Diabetic: NF 230.5 ± 1.51 205.1 ± 15.0 227.4 ± 5.61 231.4 ± 12.4NFR 228.6 ± 1.73 241.25 ± 3.5  224.1 ± 12.4 255.8 ± 9.2  p value 0.430.03* 0.82 0.03* Non- diabetic: NF 230.1 ± 2.45 272.0 ± 3.27 285.7 ±3.76 304.4 ± 3.73 NFR 224.0 ± 1.86 259.9 ± 5.74 280.6 ± 4.09 302.6 ±3.58 p value 0.07 0.10 0.38 0.73 * = significant at 95%

The RNA enriched diet (NFR) appeared to enhance body weight in thediabetic wounded rats on days 5 and 15.

EXAMPLE 8 Interpretation of Histology Examination in ExperimentalDiabetic Rats

As may be seen from the histology report that inflammatory evidence wascategorized in three types.

Gr. 1—minimal changes with some in epidermis and muscularis serosa.

Gr. 2—moderate changes just under the epidermis

Gr. 3—moderate changes throughout the skin layers.

If one assigns the rat numbers to these categories then take thepercentages then the NFR diabetic group fares slightly better than thediabetic NF group. If one looks cumulatively then the differencenarrows. However with the given conditions of the experiment, histologicobservation shown only a slight difference between the two groups. Thisis only one experiment and appropriate conditions and modifications ofthe protocol need to be done. Table 7 shows the skin pathology report.

TABLE 7 Diagnostic Laboratory Health Evaluation Histopathologicevaluation requested for 58 rat skin sections (29 H&E and 29 Movatstained sections), labeled Rat ADK 1-32 (slides 3, 14, and 19 were notpresent). Sections were evaluated for relative amounts of inflammationand fibrosis. All sections contained the epidermal, dermal andsubcuticular layers. Diabetic/Non- Skin # Diabetic NF/NFRHistopathologic Evaluation: 10 Diabetic NFR No significanthistopathologic 12 Diabetic NFR abnormalities were found. 13 DiabeticNFR 24 Non-Diabetic NF 31 Non-Diabetic NFR 32 Non-Diabetic NFR 20Non-Diabetic NF No significant histopathologic abnormalities were foundin the epidermal and dermal layers. Focal area of subcuticular musclethinning with minimal inflammation was present. 15 Diabetic NFR Smallfoci of epidermal thickening, with minimal inflammation present in thedeep dermis. The underlying subcuticular muscle is thin, and contains amild mixed inflammatory cell infiltrate 7 Diabetic NF Focal area ofsuperficial dermal fibrosis, underlying a slightly thickened epidermis.The area of fibrosis is composed of almost mature collagen, with minimalinflammation present. The deep dermis in this area contains scatteredadnexa (hair follicles) and mild mixed inflammatory cell infiltrates.Underlying subcuticular muscle is interrupted, fragmented, and containsimmature connective tissue and minimal to moderate inflammation. 8Diabetic NF Focal area of superficial dermal fibrosis, 29 Non-DiabeticNFR underlying a slightly thickened epidermis, and composed of maturingcollagen, lacking adnexa, with minimal inflammation present.Subcuticular muscle is interrupted and fragmented in slide 29, andthinned in slide 8, and contains immature connective tissue and minimalto moderate inflammation. 2 Diabetic NF Focal area of dermal flbrosis,underlying 22 Non-Diabetic NF a slightly thickened epidermis, and 23Non-Diabetic NF composed of maturing collagen, lacking 25 Non-DiabeticNFR adnexa, with mild to moderate inflammation present. Focal area ofsubcuticular muscle thinning with minimal to moderate inflammation waspresent. 1 Diabetic NF Focal area of dermal fibrosis, underlying 4Diabetic NF a slightly thickened epidermis, and 5 Diabetic NF composedof maturing collagen, lacking 9 Diabetic NFR adnexa, with minimal tomoderate 11 Diabetic NFR inflammation present. Subcuticular 16 DiabeticNFR muscle is interrupted, fragmented, and 17 Non-Diabetic NF containsimmature connective tissue and 18 Non-Diabetic NF minimal to moderateinflammation. 26 Non-Diabetic NFR 28 Non-Diabetic NFR 30 Non-DiabeticNFR 6 Diabetic NF focal area of dermal fibrosis, underlying 21Non-Diabetic NF a slightly thickened, hyperkeratotic 27 Non-Diabetic NFRepidermis with an overlying small foci of necrotic cellular debris andkeratin (scab). The dermis in this area is composed of maturingcollagen, lacking adnexa, with minimal to moderate inflammation present.Subcuticular muscle is interrupted, fragmented, and contains immatureconnective tissue and minimal to moderate inflammation.

EXAMPLE 9 Effects of Diet on Wound Healing

Wounds were created in anesthetized animals. A 4 mm punch device wasused to remove a patch of full thickness skin. Wounds were photographedon day 0, 2 and 5. (See FIGS. 2 and 3). The pictures were transferred toCD-ROM, then digitized using NIH 1.40 Image program to measure thesurface area for contraction of the wounds. After two weeks all thewounds appeared closed and the mice were photographed in group (see FIG.4).

Histology: H. & E. staining as well as Movat staining forcollagen/elastin.

NF Group:

1. Mild, multifocal infiltrates of mixed polymorphonuclear andmononuclear cells in and around the area of fibrosis.

2. Mild, diffuse infiltration of mixed inflammatory cells.

3. Moderate, multifocal, infiltration of mixed inflammatory cells withscattered microabscesses.

4. Mild, diffuse infiltration of mixed inflammatory cells.

5. Mild to moderate diffuse infiltration of mixed inflammatory cells.

NFR Group:

6. Moderate, diffuse infiltrations of mixed inflammatory cells withscattered microabscesses.

7. No area of fibrosis in sections examined.

8. No area of fibrosis in sections examined.

9. No area of fibrosis in sections examined.

10. Mild, diffuse infiltration of mixed inflammatory cells.

H. & E. observation indicates that NFR diet is more efficacious than NFdiet.

No observable differences in collagen/elastin staining was found in skinsections examined. Table 8 summarizes the surface area of the mousewounds at day 0, 2 and 5.

TABLE 8 Surface Area of Contracting Wounds in Dietary Mice on Day 0 andDay 2 and 5 Post Wounding Wound area/1 mm2, mean + sem (%) Diet Day 0Day 2 Day 5 NF 21.97 ± 1.92 16.36 ± 2.41 16.24 ± 2.41 NFR 21.37 ± 1.5315.18 ± 3.06 13.14 ± 4.1  n = 5/group

It is noted that the nutritional nucleotide addition promotes a moreaccelerated decrease in wound area.

EXAMPLE 10 Ribonucleotides from RNA Enhance Wound Healing in a ProteinStarvation Model

This example concerns the use of nucleotides in a protein starvationsetting. This would be applicable particularly to patients on dextroseiv during hospital stays in which it would be desirable to maintainimmune function.

TABLE 9 Wound Hydroxyproline Content in Protein-Starved orNucleotide-Starved or Supplemented Dietary Groups Diet Group (n = 18-20)μg OHP/cm PTFE NF 32 ± 4  PF 23 ± 2* PFR  28 ± 2** NFR 40 ± 5  *p < 0.05vs PFR, NF and NFR **p < 0.05 compared to NFR

The data demonstrate an enhancement in collagen formation even in theabsence of dietary protein. Addition of both protein and RNA isdesirable to achieve maximal healing effects. This suggests applicationof such nutritional supplementation to optimize wound healing forpatients subject to protein-free and/or a nucleotide-free nutrition. Theprotein free dietary situation may occur in dextrose iv feeding and insome disease states. A wound will clearly heal faster when RNA is addedeven in the protein free situation.

Further modifications in alternative embodiments of this invention willbe apparent to those skilled in the art in view of this description.Accordingly, this description is to be construed as illustrative onlyand is for the purpose of teaching those skilled in the art the mannerof carrying out the invention. It is to be understood that the forms ofthe invention herein shown and described are to be taken as thepresently preferred embodiments. Various changes may be made in thesemethods and compositions. For example, equivalent elements or materialsmay be substituted for those illustrated and described herein, andcertain features of invention may be utilized independently of the useof other features, all as would be apparent to one skilled in the artafter having the benefit of this description of the invention.

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What is claimed is:
 1. A method for promoting wound healing in adiabetic subject, said method comprising: preparing a dietarycomposition supplemented with one or more compound selected from thegroup consisting of adenine, uracil, inosine, and adenosine and mixturesthereof in an amount effective for promoting wound healing; and feedingsaid subject the composition.
 2. A method for promoting wound healing inan animal subject to protein deficient nutrition, said methodcomprising: preparing a dietary composition supplemented with one ormore compound selected from the group consisting of adenine, uracil,inosine, and adenosine and mixtures thereof in an amount effective forpromoting wound healing; and feeding said wounded animal with thecomposition.
 3. A method for the promotion of wound healing in asubject, said method comprising: preparing a composition supplementedwith one or more compound selected from the group consisting of adenine,uracil, inosine, and adenosine and mixtures thereof in an amounteffective for promoting wound healing; and treating said subject withthe composition such that the wound area has increased concentrations ofthe compound, wherein said treating is by enteral administration.
 4. Amethod for promotion Of wound healing in an animal, said methodcomprising: obtaining a crosslinked collagen mesh; preparing acomposition supplemented with one or more compound selected from thegroup consisting of RNA, uracil and inosine in an amount effective forpromoting wound healing; placing the crosslinked collagen mesh on thewound; and treating said wounded animal with the composition such thatthe wound area has increased concentrations of the compound.
 5. Themethod of any one of claim 1, 2, 3 or 4 wherein the amount of thecomposition is sufficient to provide a dose of about 0.0034 to about0.17 g nucleotide per kg of body weight per day.
 6. A method for thepromotion of wound healing in a subject, said method comprising:preparing a composition supplemented with one or more compound selectedfrom the group consisting of RNA, uracil and inosine in an amounteffective for promoting wound healing; and treating said subject withthe composition such that the wound area has increased concentrations ofthe compound, wherein the treating is topical.
 7. A method for thepromotion of wound healing in a subject, said method comprising:preparing a composition supplemented with one or more compound selectedfrom the group consisting of RNA, adenine, uracil, inosine, andadenosine in an amount effective for promoting wound healing; andtreating said subject with the composition such that the wound area hasincreased concentrations of the compound, wherein said treating is byintravascular administration.
 8. A method of promoting wound healing inan animal, said method comprising: preparing a composition comprising aconcentration of free pyrimidine bases, nucleosides or nucleotideseffective for the promotion of wound healing in a pharmaceuticallyacceptable carrier; and treating an animal in need thereof with atherapeutically effective concentration of the composition.
 9. Themethod of claim 8, wherein the animal is a human.
 10. The method ofclaim 8, wherein the wound is a surgical wound.
 11. The method of claim10, wherein the wound is a surgical wound and the composition isadministered to the animal as a surgery pretreatment.
 12. The method ofclaim 8, wherein the composition comprises free pyrimidine nucleotides.13. The method of claim 12, wherein the animal is treated with fromabout 0.00034 to about 0.17 g nucleotides/kg body weight/day.
 14. Themethod of claim 8, wherein the composition comprises free pyrimidinenucleosides.
 15. The method of claim 14, wherein the animal is treatedwith from about 0.00022 to about 0.12 g nucleosides/kg body weight/day.16. The method of claim 8, wherein the pyrimidine base may be selectedfrom the group consisting of uracil, thymine and cytosine.
 17. Themethod of any one of claim 1, 2, 3 or 4 wherein the amount of thecomposition is sufficient to provide a dose of about 0.00022 to about0.12 g per kg of body weight per day.
 18. A method for enhancing therate of wound healing in an animal comprising administering to an animalin need thereof a therapeutically effective concentration of pyrimidinebases, nucleosides or nucleotides or mixtures thereof formulated in apharmacologically acceptable carrier.
 19. The method of claim 18,wherein the animal is a human.
 20. The method of claim 18, whereinpyrimidine nucleotides arc administered to the animal.
 21. The method ofclaim 20, wherein from about 0.00034 to about 0.17 g nucleotides/kg bodyweight/day is administered.
 22. The method of claim 18, whereinpyrimidine nucleosides are administered to the animal.
 23. The method ofclaim 22, wherein the animal is treated with from about 0.00022 to about0.12 g nucleosides/kg body weight/day.
 24. A pretreatment method forenhancing the rate of wound healing in an animal undergoing surgerycomprising; administering a therapeutically effective concentration ofpyrimidine bases, nucleosides or nucleotides or mixtures thereof in apharmacologically acceptable carrier to an animal prior to surgery. 25.The method of claim 24, wherein the pyrimidine bases, nucleosides ornucleotides or mixtures thereof are administered to the animal at least4 weeks prior to surgery.
 26. The method of claim 24, wherein pyrimidinenucleotides are administered.
 27. The method of claim 26, wherein thenucleotides are administered in a concentration of about 0.00034 toabout 0.17 g/kg body weight/day.
 28. The method of claim 24, whereinpyrinidine nucleosides are administered.
 29. The method of claim 28,wherein the nucleosides are administered in a concentration of about0.00022 to about 0.12 g/kg body weight/day.
 30. A method of enhancingcollagen concentration at a wound site in an animal comprisingadministering a preparation of pyrimidine bases, nucleosides ornucleotides in a pharmacologically acceptable carrier to the animal. 31.A pretreatment method for enhancing the rate of wound healing in ananimal undergoing surgery comprising; administering a compositioncomprising pyrimidine bases, nucleosides or nucleotides or mixturesthereof in a pharmacologically acceptable carrier to an animal prior tosurgery.